Identification and characterization of gut-associated lactic acid bacteria isolated from the bean bug, Riptortus pedestris (Hemiptera: Alydidae)

Lactic acid bacteria (LAB) are beneficial bacteria for humans and animals. However, the characteristics and functions of LAB in insects remain unclear. Here, we isolated LAB from the gut of Riptortus pedestris, a pest that is a significant problem in soybean cultivation in Korea, and identified two Lactococcus lactis and one Enterococcus faecalis using matrix-associated laser desorption/ionization-time of flight and 16S rRNA analyses. All three LAB strains survived at pH 8, and L. lactis B103 and E. faecalis B105 survived at pH 9 for 24 h. In addition, these strains survived well in simulated gastric juice of humans containing pepsin and exhibited high resistance to bile salts. Two strains of L. lactis and one of E. faecalis maintained constant density (> 104 colony-forming units [CFU]/mL) at pH 2.5, but viability at pH 2.2 was strain-dependent. The three LAB were reinoculated into second-instar nymphs of R. pedestris and colonized well, reaching a constant density (> 105 CFU/gut) in the adult insect gut. Interestingly, feeding of these LAB increased the survival rate of insects compared to the negative control, with the largest increase seen for L. lactis B103. However, the LAB did not increase the weight or length of adult insects. These results indicate that insect-derived LAB possess the traits required for survival under gastrointestinal conditions and have beneficial effects on insect hosts. The LAB infection frequency of the wild bean bug populations was 89% (n = 18) in Gyeongsangnam-do, South Korea. These LAB can be utilized as a novel probiotic in the cultivation of beneficial insects. This study provides fundamental information about the symbiosis between insects and LAB, and a novel concept for pest control.


Introduction
Insects are the most diverse group of animals in terms of species number and have the largest biomass in almost all environments on earth [1,2]. The diversification of insects is closely 7.0 (PB). The guts were homogenized with a plastic pestle and serially diluted with PB. The diluted gut solution was spread onto de Man, Rogosa, and Sharpe (MRS) agar medium (Difco, Detroit, MI, USA) and incubated at 28˚C for 3 days in a CO 2 incubator (Thermo Scientific, Waltham, MA, USA). Bacterial colonies that were uniform, round, and white were selected, purified, and stored at -80˚C in MRS broth mixed with glycerol (25%, v/v).

Identification of LAB
To confirm the identity of bacterial isolates (B103-B105), whole-cell matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry using the program MALDI Biotyper v. 3.0 (Bruker Daltonics, Bremen, Germany) were performed according to the manufacturer's instructions (Bruker Daltonics). Bacterial samples for MALDI-TOF MS were prepared as previously described [26]. Mass spectra were analyzed using a microflex LT mass spectrometer (Bruker Daltonics) and default parameters (positive linear mode; laser frequency, 60 Hz; ion source 1voltage, 20 kV; ion source 2voltage, 16.7 kV; lens voltage, 7.0 kV; and mass range, 2 kDa to 20 kDa). For each spectrum, 240 laser shots in 40-shot steps from different positions of the sample spot were accumulated and analyzed (automatic mode, default settings) as previously described [26]. MALDI-TOF MS data were interpreted according to the manufacturer's instructions (Bruker Daltonics). Scores of �2.0 are considered reliable for the species level, and scores of �1.7 but <2.0 are acceptable for the genus level, and scores below 1.7 are considered unreliable [26]. The partial 16S rRNA region was amplified with the primers 27mF (5 0 -AGAGTTTGATCMT GGCTCAG-3 0 ) and 1492mR (5 0 -GGYTACCTTGTTACGACTT-3 0 ) [27]. Total DNA extraction was conducted as described by Choi et al. (2019) [28]. Polymerase chain reaction (PCR) was performed on a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA) using PCR premix (Bioneer, Daejeon, Korea), 10 ng genomic DNA, and 1 mM of each primer with a program of 98˚C for 30 s, 55˚C for 30 s, and 70˚C for 1 min, followed by a final 4-min extension at 72˚C. The PCR product was confirmed using 0.8% agarose gel electrophoresis and purified using the Expin Gel SV kit (GeneAll Biotechnology, Seoul, Korea). Sequencing was performed by Macrogen's sequencing service (Macrogen, Daejeon, Korea). The resulting DNA sequences were analyzed using the National Center for Biotechnology Information GenBank database (https://www.ncbi.nlm.nih.gov/blast/). A phylogenetic tree based on the nucleotide sequences of the 16S rRNA gene was generated using MEGA 7.0 software and the neighbor-joining method [29]. Streptococcus thermophilus LMG 18311 was used as an outgroup because it is phylogenetically closely related to the genera Lactococcus and Enterococcus [30].

Survival of LAB at alkaline pH
Survival of LAB strains at weak alkaline pH was tested. MRS broth was adjusted to pH 8 or 9 using 1 N NaOH. MRS broth adjusted to pH 7 was used as a control. Cells from 2-day-old cultures of the three isolates grown at 28˚C were inoculated into pH-adjusted MRS broth at a concentration of approximately 10 9 colony-forming units (CFU)/mL and incubated at 28˚C for 24 h. Samples were taken at 6 h intervals, serially diluted, and spread onto MRS medium. The plates were incubated at 28˚C for 3 days. The viable cell population was determined by colony counting.

Survival of LAB in simulated gastric juice and bile salt solution of humans
Simulated gastric juice of humans was prepared by adding 1% pepsin to MRS broth adjusted to pH 2.2 or 2.5 using 1 N HCl, according to a previously reported method [31]. Cells from 2-day-old cultures of the three isolates grown at 28˚C were inoculated into simulated gastric juices at a concentration of approximately 10 9 colony-forming units (CFU)/mL and incubated at 28˚C for 2 h. Samples were taken at 30 min intervals, serially diluted, and spread onto MRS medium. The plates were incubated at 28˚C for 3 days. The viable cell population was determined by colony counting.
For the bile tolerance assay, cells from 2-day-old cultures of the three isolates grown at 28˚C were inoculated into MRS broth containing 0.1 or 0.5% (w/v) bile salt (oxgall) (Difco) at a concentration of approximately 10 9 CFU/mL and then incubated at 28˚C for 24 h according to a previously reported method [32]. Samples were taken at 6 h intervals, serially diluted, and spread onto MRS medium. The plates were incubated at 28˚C for 3 days. The viable cell population was determined through colony counting.

Determination of glucose utilization and pH values
To assess the glucose utilization of the three isolates, a D-glucose assay kit (glucose oxidase/ peroxidase [GOPOD]; Megazyme, Wicklow, Ireland) was used according to the manufacturer's instructions [33]. The three isolates were grown on MRS broth at 28˚C for 3 days and sampled at 12-h intervals; the supernatants were collected. Then, 3 mL GOPOD reagent was added to each 100-μL aliquot of supernatant and incubated at 42˚C for 20 min. Distilled water was used as the reagent blank solution. After the color reaction, absorbance of the samples was read at 510 nm using a spectrophotometer (Genesys 6; Thermo Electron Corp., Waltham, MA, USA). The pH of the MRS culture medium of the isolates was measured at 4-h or 24-h intervals for 3 days using a pH meter (SevenEasy; Mettler-Toledo, Greifensee, Switzerland).

Analysis of organic acids
To determine whether the three isolates use glucose as a carbon source for conversion to lactic acid, the isolates were cultured in MRS broth containing 2% glucose at 28˚C for 2 days. The bacterial cultures were centrifuged and the supernatant were collected. Each supernatant was filtered through a 0.45-μm cellulose acetate filter for high-performance liquid chromatography (HPLC) analysis. The concentrations of organic acids, such as lactic acid and acetic acid, were determined through HPLC (L-2200; Hitachi, Tokyo, Japan) using an ultraviolet detector (L-2400; Hitachi) and a column (Metacarb 87H; Varian, Palo Alto, CA, USA). HPLC profiles of the culture filtrates were analyzed through comparison with the elution profiles of standard organic acids injected separately.

Bean bug feeding and evaluation of LAB survival in the gut
A bean bug feeding trial was conducted to investigate the survival of LAB in the gut. Rifampicin, a bacterial RNA polymerase inhibitor, was previously used for bacterial colonization monitoring studies in bean bugs [34]. Spontaneous rifampicin-resistant strains of LAB were obtained through sequential selection on MRS agar containing rifampicin starting at 5 μg/mL and up to 25 μg/mL. Spontaneous rifampicin-resistant strains of LAB were cultured on MRS containing 25 μg/mL rifampicin at 28˚C for 2 days, and the cultured cells were harvested, washed with DWA, and suspended in DWA at a concentration of approximately 10 8 CFU/ mL. Newly molted second-instar nymphs were not supplied with DWA for 24 h, and the thirsty nymphs were then supplied with DWA containing LAB cells for 24 h. After inoculation, the nymphs were reared as described above. At 21 days post-inoculation, five adult bean bugs were dissected and their guts were transferred to a tube containing 500 μL PB. The guts were completely homogenized with a plastic pestle and serially diluted with PB. The diluted solution was spread onto MRS agar containing 25 μg/mL rifampicin. After 3 days of incubation at 28˚C, colonies on the plates were counted.

Evaluation of R. pedestris survival rate, weight, and length
To assess the survival rate of insects after feeding of LAB cells to second instar nymphs, dead insects were counted daily for 20 days until the nymphs became adults. At 21 days after bacterial inoculation, adults of R. pedestris were immersed in acetone for 5 min, and completely dried at 70˚C in an oven; their dry body weight was then measured. The insects were not fed for 24 h before killing. Body length was measured from the head to the tip of the abdomen. Mean differences are shown on Cumming estimation plots [35] as dots; 95% confidence intervals are indicated by the ends of the vertical bars. All estimation plots were generated using https://www.estimationstats.com/#/.

Natural LAB infection in the wild population of bean bug
To determine the natural LAB infection rate of wild bean bug populations, R. pedestris adults were collected from soybean fields located in 18 regions of Gyeongsangnam-do, South Korea during 2021-2022 (one per region). LAB isolation was performed according to the method described above. Among the colonies formed on MRS after culturing in a CO 2 incubator, the identical colonies having the same color and shape as L. lactis and E, faecalis were purely isolated and identified using the MALDI-TOF MS utility.

Identification of LAB in the gut of Riptortus pedestris
To isolate the LAB associated with the bean bug gut, bacteria were isolated from the guts of healthy adult R. pedestris. Gram-positive, oxidase-negative bacteria with round, white colonies were consistently recovered on MRS medium containing 2% glucose. Three bacterial isolates (labeled B103-B105) were selected for identification and characterization.
MALDI-TOF mass spectrometric analysis revealed that B103 and B104 were Lactococcus lactis with scores of 2.236 and 2.256 (score �2.0 = identification to the species level [26]), respectively, while B105 yielded score of 2.331 as Enterococcus faecalis.
16S rRNA gene sequence analysis revealed that two isolates (B103 and B104) had identical 16S rRNA gene sequences. BLAST search confirmed that the 16S rRNA gene sequences of B103 and B104 (1,406 bp; GenBank accession nos. ON834457 and ON834458) shared 100% similarity to Lactococcus lactis 1700 (MT597572) isolated in China, and the 16S rRNA gene sequence of B105 (1,401 bp; GenBank accession no. ON834462) shared 100% similarity to Enterococcus faecalis ABCW (ON564563) obtained from cheese whey in India. In phylogenetic analysis, B103 and B104 clustered in the same group as Lactococcus lactis, while B105 clustered in the same group as Enterococcus faecalis (Fig 1).

Survival of LAB at weak alkaline pH
In most insects, the digestive juices in the midgut generally fall within the pH range of 6-8 [36][37][38]. To evaluate the survival of LAB under weak alkaline conditions, we assessed the survival of three LAB strains in MRS broth at pH 8 and 9 for 24 h. The viable cell counts of the three LAB strains at pH 7, used as a control, did not change over 24 h, and were similar even at pH 8 (Fig 2). L. lactis B103 decreased slowly by 6.83 log10 CFU/mL over 24 h of exposure to pH 9 (Fig 2A), while the survival of L. lactis B104 decreased sharply after 24 h of exposure to pH 9, down to 2.63 log10 CFU/mL (Fig 2B). These results indicate that all tested strains are resistant to the simulated insect gut pH environment and that species specificity appears among LAB strains even within a species.

Survival of LAB in simulated gastric juice and bile salt solution of humans
We next assessed the viability of LAB in a low pH environment simulating the human gut to determine critical probiotic availability. To evaluate the survival of LAB under acidic conditions, we assessed the survival of three LAB strains in simulated gastric juice (pH 2.2 or 2.5) containing 1% pepsin for 120 min. E. faecalis B105 exhibited the highest survival rate over 120 min at pH 2.2, while the survival of L. lactis B104 decreased sharply after 60 min of exposure, with a reduction of 4.26 log10 CFU/mL seen at 120 min. B103, a second L. lactis strain, decreased slowly, by 4.78 log10 CFU/mL over 120 min (Fig 3A). These results indicate that the survival rate differs among LAB strains, even within a species. L. lactis B103, L. lactis B104, and E. faecalis B105 all had high survival rates at pH 2.5 over 120 min of exposure (Fig 3B).
These results indicate that all tested strains exhibit resistance to the gastric juice environment.
To evaluate whether bile salts reduce the survival of LAB, the three LAB strains were assessed for survival in MRS media containing 0.1% and 0.5% bile salt (oxgall). All tested LAB isolates showed high survival rates after 24 h of exposure to 0.1% and 0.5% oxgall (Fig 4A and  4B). These results indicate that the three LAB strains have significant tolerance to bile salts.

Glucose utilization of LAB and pH change
To investigate glucose utilization by LAB and the associated pH change, glucose consumption of the three LAB strains was measured, as well as the pH of MRS medium. E. faecalis B105 grew rapidly, reaching the early stationary phase after 12 h in MRS medium, and its cell density led to an optical density at 600 nm (OD 600 ) value of 1.69 after 72 h. In contrast, L. lactis B104 grew slowly and reached a low cell density of OD 600 = 0.99 after 72 h, while B103, which is the same species as M104, had the lowest cell density of OD 600 = 0.76 after 72 h (Fig 5A). E. faecalis B105 consumed glucose rapidly and had consumed 65% of the supplied glucose over 72 h, and its culture pH decreased rapidly to 4.4 over 24 h. In contrast, L. lactis B103 and B104 consumed 43% and 50% of the available glucose over 72 h, and reached pH of 5.0 and 4.8 over 24 h, respectively (Fig 5B and 5C). These results indicate that E. faecalis B105 utilizes glucose well and decreases pH more effectively than the other two isolates, consistent with the growth rates observed in MRS.

Organic acid production by LAB from glucose
Organic acid production by LAB strains was investigated in MRS medium containing 20 g/L of glucose over 48 h. As shown in Fig 6, E. faecalis B105 exhibited a significant difference compared to the other strains, achieving higher contents of acetate, lactate, and propionate. L. lactis strain B104 produced significantly less acetate, lactate, and propionate than B105, but significantly more acetate and lactate than B103 strain. In contrast, B103 produced low contents of lactate and propionate; no acetate was detected. These results indicate that E. faecalis B105 efficiently utilized glucose for organic acid production. Organic acid production from glucose, differed among strains, even within the species L. lactis.

Colonization of R. pedestris gut by LAB
To confirm whether the inoculated LAB efficiently colonizes the gut of R. pedestris, the number of bacteria per gut of adult insects that were inoculated at second-instar nymphs was investigated through bacterial colony counting. The bacterial population abundances of L. lactis B103 and B104, and E. faecalis B105 were 1.21 × 10 7 , 5.27 × 10 5 , and 3.24 × 10 6 CFU/ gut, respectively (Fig 7). These results indicate that the three tested LAB strains proliferated and successfully colonized the gut of insects to a consistent density after inoculation via feeding.

Effect of LAB on the survival and growth of R. pedestris
Next, to determine the impacts of the three LAB strains on the survival and growth of R. pedestris, the survival rate, length, and weight of experimentally inoculated adult insects were measured. The survival percentages of insects inoculated with L. lactis B103 and B104, and E. faecalis B105 were 78.9, 60.5, and 55%, respectively (Fig 8A). In contrast, the DWA treatment showed a survival rate of 47.3%. However, insects inoculated with the three LAB strains showed no difference in body weight or length compared to the DWA treatment (Fig 8B and  8C). These results indicate that the three LAB strains improve the viability of insects but have no growth-promoting effect. For presentation, all data were normalized to cell population (log10 CFU/mL). Columns marked with an asterisk (*) indicate statistically significant differences for acetate production between B104 and B105 strains using Student's t test (P � 0.05). nd: not detected. One-way ANOVA, Tukey test, P < 0.05 for lactate and propionate of the three LAB strains. Different letters indicate significant differences (P < 0.05) between LAB strains. https://doi.org/10.1371/journal.pone.0281121.g006

Natural LAB infection in the wild population of bean bug
LAB were isolated according to the method described above and identified using MALDI-TOF MS identification (score �2.0). The LAB infection frequency of the wild bean bug populations was 89% (16/18) (Fig 9). Two cultivable LAB, L. lactis and E. faecalis, were isolated from a single bean bug, but not simultaneously (Fig 9). These results indicate that the LAB-bean bug symbiosis depends on the habitat environment. The 16 LAB isolates obtained from the guts of bean bugs and their collection regions are listed in Table 1.

Discussion
The bean bug, R. pedestris, a problematic pest in soybean cultivation fields, was easily collected, and LAB were isolated from their guts. However, few species of bacteria were isolated, and the number of LAB was low. We isolated two strains of Lactococcus lactis and one of Enterococcus faecalis (Fig 1). The pH condition of the insect gut is somewhat alkaline, which may limit the growth and abundance of LAB [2]. In addition, the results impacted by LAB comprising only a small proportion of the total microflora [39,40].
L. lactis is mainly isolated from plants and the environment.
This LAB is used for cheese production and fermentation of foods including vegetables, meat, and wine [41,42]. In addition, this bacterium has been genetically modified for the treatment of human diseases [43]. L. lactis has GRAS status and is beneficial to humans [44]. L. lactis has been isolated as a symbiotic bacterium from the oriental fruit fly and reported to affect the development, morphology, and survival of insects [45]. L. lactis was identified in mealworms (Tenebrio molitor) by 16S amplicon sequencing [46]. The LAB E. faecalis is isolated mainly from the guts of healthy humans and many animals. However, this species has also been reported as an opportunistic pathogen that produces bacteriocins and is highly resistant to antibiotics [47][48][49]. E. faecalis has been isolated from the feces of houseflies and cockroaches and reported as a symbiotic bacterium in the gut of the African cotton leafworm (Spodoptera littoralis) [50][51][52]. E. faecalis was also identified in mealworms (Tenebrio monitor) via 16S amplicon sequencing [46]. Some strains of this species are pathogenic to the larvae of the greater wax moth (Galleia mellonella) and African cotton leafworm (S. littoralis) [49,51].
We focused on whether B103-B105 isolated from the bean bug can function as LAB. LAB benefit host cells only if they survive low pH conditions (pH 2.0-2.5) and toxic bile salts during passage through the gastrointestinal tract and adhere well to intestinal cells [52,53]. The three isolates investigated in this study survived at densities of > 4.2 log10 CFU/mL in simulated gastric juice at pH 2.2 (Fig 3A). B103 and B104 belong to the same species of L. lactis, but showed different sensitivities to low pH. These results were consistent with previous research, which indicates that strains of the same Lactobacillus species may exhibit differences in viability at low pH and strain-dependent survival [52,54]. All three isolates survived well in simulated gastric juice at pH 2.5 or 0.5% bile salt solution (Figs 3B and 4). All isolates produced organic acids, including lactic acid using glucose, and could lower the surrounding pH (Fig 6). Therefore, we suggest that these three isolates obtained from the gut of the bean bug exhibit the critical characteristics of probiotics. According to the viability assay in the simulated insect gut pH, i.e. weakly alkaline environment, all three strains survived well in MRS broth at pH 8, and L. lactis B103 and E. faecalis B105 survived at pH 9 except for L. lactis B104 (Fig 2). Colonization ability in the intestine is another important trait of LAB. B103-105 were confirmed to colonize the gut of adult insects at densities 10 5 -10 7 CFU/gut (Fig 7). These results indicate  that bacteria fed to second-instar nymphs proliferated and successfully colonized the gut, while the larvae molted into adults. In a previous study, the Caballeronia symbiont explicitly localized in the midgut crypts (M4) was monitored for localization using fluorescence labeling techniques [55]. In this study, we showed the total colony population of LAB instead of LAB localization in the gut of bean bugs, as it is currently unknown whether LAB are restricted to specific gut sections. Interestingly, these follow-up studies on this issue will have implications for understanding the natural ecology of LAB and will provide new opportunities for industrial strain development.
The Caballeronia symbiont has been reported as a specific symbiotic bacterium of bean bugs that colonizes the gut at high density, increases viability during the molting process and improves the growth of adults [56,57]. In this study, inoculation with LAB also improved the viability of insects. However, no effect was observed on the length or weight of the insects (Fig  8). These results suggest that the three LAB strains tested here colonize the gut to a constant density and improve the detrimental physiological or biochemical alterations that occur during molting, allowing the host to survive well during the initial molting process. Additionally, these strains may benefit the host by supplying nutrients, such as organic acids and vitamins produced during fermentation. Bacteria coexisting with various insects, such as the fruit fly, African cotton stainer, beetle, and stinkbug, play beneficial roles in processes such as nutrition acquisition, vitamin supply, immunity, detoxification, and the acquisition of insecticide resistance [11,[58][59][60]. However, the symbiotic LAB-bean bug relationship is not host-specific as in the Caballeronia-bean bug symbiosis. The LAB species, including L. lactis and E. faecalis, have already been discovered in other insects. Depending on the insect species, their roles in beneficial or harmful relationships have been studied differently [44][45][46][47][48][49][50][51]. Moreover, LAB constitute a very small portion of the total microflora of insects [39,40]. The regional differences in LAB infection found in the wild populations of bean bugs support that the symbiosis between L. lactis or E. faecalis and bean bug is not host-specific as in the Caballeronia-bean bug symbiosis but depends on the habitat environment. In addition, if methods such as fluorescent protein tagging or fluorescent in situ hybridization are applied to visualization LAB colonies in the insect gut, it will be helpful to understand the symbiosis of LAB-bean bug and will be necessary for follow-up studies.
The midgut of R. pedestris consists of four sections: M1, M2, M3, and M4, and the Caballeronia symbiont specifically colonizes the M4 section. Itoh et al. [34] demonstrated that in a single inoculation with taxonomically distinct bacteria, Burkholderia fungorum and Pandoraea norimbergensis colonized M4; however, other tested species, including Enterococcus hirae and Lactobacillus casei, showed little or no infection in M4. In co-infection, the symbiotic relationship between bean bugs and the Caballeronia symbiont is achieved by competition between gut bacteria in M4, and the Caballeronia symbiont consistently outperformed B. fungorum and P. norimbergensis in M4. It is currently unknown whether the LAB in this study will colonize the M4 section when co-infected with the Caballeronia symbiont, but results from previous reports suggest that it is unlikely that the LAB will colonize the M4 section. However, the results of LAB colonization in the guts and improvement of survival rate suggest a beneficial effect on insects. Further research is needed as the results on this issue will be valuable for future probiotics applications.
In this study, we isolated and identified L. lactis and E. faecalis from the bean bug R. pedestris, pest that causes severe damage to legumes in Korea, and demonstrated that these bacteria have the essential features of LAB. The present study is the first to report LAB isolated from the bean bug with potential application as probiotics. This study provides fundamental information about the symbiosis between insects and LAB, and a novel concept for pest control.